Extracellular nucleotides (e.g. ATP, ADP, UTP) provide important environmental signals to platelets and cells, that areoperative within the vasculature. These mediators activate type-2 purinergic/ pyrimidinergic (P2Y and P2X) receptors on platelets, endothelium, vascular smooth muscle, and leukocytes. This process modulates platelet activation andadhesion, cellular metabolism, nitric oxide (NO) release, endothelial activation, proliferation and apoptosis. Ectonucleotidases hydrolyze extra-cellular nucleotides, ultimately to the respective nucleosides. Members of the CD39 family of such ectoenzymes appear to be differentially expressed at high levels within the vasculature. Endothelial cells (EC) and vascular smooth muscle cells (VSMC) express CD39/NTPDase1, which has both ecto-ATPase and -ADPase activities, while pericytes are associated with CD39L(ike)1/NTPDase2, a preferential ecto-ATPase. Global deletion of cd39 in mice results in hemostatic defects, inflammatory changes with thromboregulatory disturbances that perturb vascular homeostasis and preclude long term transplant graft survival. As these mutant mice unexpectedly exhibit insulin resistance, actions of insulin (and other growth factors) may be also modulated by extracellular nucleotides. This application addresses the possibility that the differential expression of CD39 and CD39L1 by the vasculature has important consequences for the temporal and spatial modulation of P2-mediated platelet and vascular cellular reactions. We propose that at sites of vascular inflammation, NTPDases may regulate thrombogenesis or vascular remodeling and modulate the local "metabolic milieu" e.g. in reperfused organs, injured vessels or vascularized grafts. SPECIFIC AIM 1: Investigate how CD39/NTPDase1 and CD39L1/NTPDase2 influence acute and chronic vascular injury by studying the acute development of arterial thrombi by confocal microscopy using mutant mice null for cd39 and/or cd39L1. We will also study how more protracted VSMC responses to vascular wall injury are altered by these vascular NTPDases in vivo. SPECIFIC AIM 2: Determine the mechanisms whereby CD39/NTPDase1 modulates acute vascular injury and graft survival. We will examine how CD39 expression both directly and indirectly influences cell activation/apoptosis with insulin responsiveness in EC and VSMC to explore the relevance of this phenomenon. SPECIFIC AIM 3: Evaluate how NTPDases interact with endothelial nitric oxide synthases (eNOS). We will determine whether effects of NTPDases and NO are additive or synergistic by regulating each in wild type and mutant mice, deficient in cd39 and/or eNOS, in the experimental models proposed above. An understanding of the respective functions of the two major vascular NTPDases and eNOS should provide insights into mechanisms of acute vascular injury and localized vasculopathy. Information gleaned from these studies may direct new therapeutic strategies for inflammatory vascular disorders, including atherosclerotic cardiovascular disease.